1. Field of Invention
The present invention relates to biotechnology and the microbiological industry, and specifically to a method for constructing an operon containing translationally coupled genes, a method for producing useful metabolites using bacteria containing the coupled genes, and a method for monitoring gene expression.
2. Description of the Related Art
Translation of genes in bacteria can be optimized using translational coupling, which is known to occur in prokaryotic cells.
It was found that E. coli trpE polar mutations were 10 times more polar for trpD gene expression than for downstream (trpC, B, or A) gene expression. This effect was shown to be the result of “translational coupling,” in which efficient translation of the trpE-trpD intercistronic region utilizes overlapping stop and start codons. Therefore, the trpE and trpD gene products form a functional complex in the cell (Oppenheim D. S, and Yanofsky C., Genetics 95(4):785-95 (1980)).
Precise frameshift and nonsense mutations were introduced into the region preceding the galactokinase gene (galK) of E. coli. These mutations alter the upstream translation termination position relative to the galK translation initiation signal. Constructions were characterized that allow ribosomes to stop selectablely before, within, or downstream from the galK initiation signal. The effects of these mutations on galK expression were monitored. Galactokinase levels are highest when upstream translation terminates within the galK initiation region. In contrast, when translation stops either upstream or downstream from the galK start site, galK expression is drastically reduced. These results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon, that is, galT, and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the galK start site (Schumperli D. et al, Cell, 30(3):865-71 (1982)).
The conditions necessary for high-level expression of methionyl bovine growth hormone (Met-bGH) in E. coli were investigated. Plasmids were constructed that contain a thermoinducible runaway replicon, ribosome binding sites which served as transcriptional and translational initiation sites for the expression of the bGH gene, and either the E. coli tryptophan or lipoprotein promoter. The expression of Met-bGH was low with either promoter. However, expression levels of up to 30% of total cell protein were obtained after the introduction of additional codons positioned 3′ to the initiating AUG codon, thus altering the NH2-terminal amino acid sequence of bGH. To obtain high-level expression of Met-bGH, a two-cistron system was constructed in which the codons that enhanced the expression of bGH were incorporated into the first cistron, and the coding region for Met-bGH was incorporated into the second cistron. This approach may be generally applicable to achieving high-level expression of a gene that contains NH2-terminal sequences that inhibit efficient expression. Analyses of the stabilities of the bGH derivatives and their transcripts in vivo suggested that the variations in the level of expression were due to variations in the efficiency of mRNA translation (Schoner B. E. et. al., Proc. Natl. Acad. Sci. USA, 81(17): 5403-7 (1984)).
Expression of trpB and trpA on the E. coli tryptophan operon is shown to be “translationally coupled”, i.e., efficient translation of the trpA coding region is dependent on the prior translation of the trpB coding region, and termination of translation at the trpB stop codon. To examine the dependence of trpA expression on the ribosome binding site sequence in the distal segment of trpB, deletion mutants of the trpB sequence were produced. Analysis of trpA expression in these deletion mutants established that the ribosome binding site sequence is required for efficient translation of the trpA segment of the trp mRNA. The translational effect on independent initiation at the trpA ribosome binding site was modest (Das A. and Yanofsky C., Nucleic Acids Res., 12 (11):4757-68 (1984)).
A trp-lac fusion system in which part of the trpA gene is fused to the lacZ gene was used to investigate whether translational coupling occurs between the tryptophan operon trpB and trpA genes in E. coli. This fusion protein has the translation initiation site of trpA but retains beta-galactosidase activity. A frameshift mutation was introduced early in trpB and its effect on transcription and translation of the trp-lac fusion was measured. The mutation resulted in a 10-fold drop in beta-galactosidase activity, but only a 2-fold drop in lacZ mRNA or galactoside transacetylase levels. An rho mutation restored the lacZ mRNA and transacetylase levels to those of the control but only increased the beta-galactosidase level to 20% of the control. These results demonstrate that if the trpB gene is not translated, efficient translation of the trpA′-lac′Z mRNA does not occur, and thus, that these genes are translationally coupled (Aksoy S. et al, J. Bacteriol., 157(2):363-7 (1984)).
The trpB and trpA coding regions of the polycistronic trp mRNA of E. coli are separated by overlapping translational stop and start codons. Efficient translation of the trpA coding region is subject to translational coupling, i.e., maximum trpA expression is dependent on prior translation of the trpB coding region. Previous studies have demonstrated that the trpA Shine-Dalgarno sequence (within trpB) and/or the location of the trpB stop codon influenced trpA expression. To specifically examine the effect of the location of the stop codon, plasmids were constructed in which different nucleotide sequences preceding the trpA start codon were retained, and only the reading frame was changed. When trpB translation proceeded in the wild-type reading frame and terminated at the normal trpB stop codon, trpA polypeptide levels increased as compared to the levels observed when translation stopped before or after the natural trpB stop codon. The proximity of the trpB stop codon to the trpA start codon therefore markedly influences trpA expression (Das A. and Yanofsky C., Nucleic Acids Res., 17(22):9333-40 (1989)).
A TGATG vector system was developed that allows for the construction of hybrid operons with partially overlapping genes, and which utilize translational coupling to optimize expression of the cloned cistrons in E. coli. In this vector system (plasmid pPR-TGATG-1), the coding region of a foreign gene is attached to the ATG codon on the vector to form a hybrid operon which is transcribed from the phage lambda PR promoter. The cloned gene is the distal cistron of this hybrid operon (‘overlappon’). The efficiently translated cro′-cat′-′trpE hybrid cistron is proximal to the promoter. The coding region of this artificial fused cistron (the length of the corresponding open reading frame is about 120 amino acids (aa)), and includes the following: the N-terminal portions of phage lambda Cro protein (20 aa), the CAT protein of E. coli (72 aa), and 3′ C-terminal codons of the E. coli trpE gene product. At the 3′-end of the cro′-cat′-′trpE fused cistron there is a region for efficient translation reinitiation: a Shine-Dalgarno sequence of the E. coli trpD gene and the overlapping stop and start codons (TGATG). In this sequence, the last G is the first nucleotide of the unique SacI-recognition site (GAGCT decreases C), and so integration of the structural part of the foreign gene into the vector plasmid may be performed using blunt-end DNA linking after the treatment of pPR-TGATG-1 with SacI and E. coli DNA polymerase I or its Klenow fragment (Mashko et. al., Gene, 88(1):121-6 (1990)).
But currently, there have been no reports of using a bacterium having an operon containing translationally coupled genes for the purpose of producing useful metabolites. A bacterium having translationally coupled genes can be also used to monitor the expression of the genes.